Components for Animal Feed and Use Thereof

ABSTRACT

A fermented liquid feed composition for a target animal is provided, the feed composition being prepared by the fermentation of a feed substrate with a lactic acid producing bacteria, the lactic acid bacteria being characterised by:
         a) being viable under the conditions prevailing in the gastrointestinal tract of the target animal;   b) being an aggregating bacteria and/or co-aggregating with one or more pathogens; and   c) being able to produce upon fermentation in the feed substrate lactic acid in an amount of at least a minimum inhibitory concentration of lactic acid.       

     A method of producing a fermented liquid feed composition is also provided. Preferred lactic acid producing bacteria for use in the composition and method are  Lactobacillus.

The present invention relates to feed materials for animals, to theprovision of fermented animal feed products, methods for theirpreparation and the use thereof.

It is common practice to prepare animal feeds as dry compositions, thatis up to 15% moisture, to avoid spoilage. The dry feed material may bestored for extended periods of time and transported with little or nodegradation. However, the cost of preparing dried feed is increasing. Inparticular, in excess of 60% of the energy costs on preparing the dryfeed are consumed in the actual drying stage. Accordingly, there is agrowing need for an alternative to the known dry animal feeds.

One such alternative are moist or liquid feeds. In this regard, moistfeeds, such as those fed to chickens and other poultry, contain up to30% by weight of water. Liquid feeds, such as those fed to pigs, containup to 70% by weight of water. Known for some time, liquid feeds can bedifficult to formulate, prepare and store in a cost effective manner. Inaddition, moist or liquid feeds are difficult to store and transportover long distances. In particular, wet or liquid animal feeds are veryprone to spoiling due to the growth of mould, bacteria and yeast, makingthe long term storage of wet and liquid animal feeds a difficultprospect. There is therefore a need to address the problem of storingand transporting wet and liquid feeds. It would also be mostadvantageous if the range of starting materials for preparing wet andliquid animal feeds could be extended. Currently, co-products andresidues from industries such as dairy, bakery and distilling are usedto prepare wet and liquid feed for animals. Raw materials withsignificant future potential are the co-products of bioethanal andbiofuel production. These are already incorporated into diets followingdrying, which is an extremely energy demanding mode of treatment. Itwould be beneficial, if a way can be found to formulate theseco-products into a moist or liquid feed. Other potential sources for rawmaterials for incorporation into moist and liquid animal feeds includehuman food grade residues from the slaughter process and from meat andfish processing. The value of much of this material is currently lostdue to poor storage, drying or disposal to landfill. All these processeshave a high energy demand and adverse environmental impact.

One improvement to the preparation of wet and liquid animal feeds is theinoculation of the feed raw material with one or more suitablemicroorganisms, to produce a so-called ‘fermented feed’. This process issynonymous with the process of ‘ensiling’, which is the ubiquitousmethod used for the preservation of herbage for feeding to ruminantanimals. The inoculant is selected to inhibit the growth of mould,yeasts and spoilage bacteria that will propagate in and spoil the feedmaterial. The process of producing fermented feed can be seen as a formof ‘biopreservation’. EP 0 906 952 discloses a bacterial strain for theensiling of straw fodder. The strain, of the genus Lactococcus, wasfound to be effective in inhibiting the growth or yeast, clostridia,mould, gram positive bacteria and certain gram negative bacteria in theensiling of green fodder.

Further, US 2002/0054935 is concerned with a livestock feed compositionsuitable for the fattening of livestock, such as cattle, goats, sheep,swine and fowl. The nutritional value of the livestock feed is increasedby inoculation with one or more strains of Aspergillus. The livestockfeed treated in this way consists of a fibrous feed material, a cereal,and an organic waste material. It appears that the Aspergillus is usedto modify the nutrient content of the feed. However, this can havedisadvantageous results, as many Aspergillus spp. produce mycotoxinsharmful to many animals.

U.S. Pat. No. 6,403,084 is concerned with mixed cultures for improvedfermentation and aerobic stability of silage. The problem of aerobicinstability of silage is addressed, in particular the rapid growth ofyeast and mould that can occur, resulting in the silage being spoiled.Further, it is noted that silage may be spoiled by the growth of yeast,even when inoculated and subjected to a good fermentation phase, inwhich microorganisms are used to ferment the silage and produce lacticacid, reducing the pH and giving rise to acid conditions. Acid-tolerantyeasts are considered to be responsible for the spoilage of fermentedsilage. As a solution to these problems, U.S. Pat. No. 6,403,084proposes inoculating the silage with a combination of thehomofermentative lactic acid bacteria Lactobacillus plantarum and theheterofermentative lactic acid bacteria Lactobacillus buchneri orLactobacillus brevis. The aforementioned combination of microorganismsis alleged to provide sufficiently low pH conditions to preserve thesilage and prevent spoiling due to the growth of mould and yeast.

WO 99/18188 describes a feed product for horses. The feed productcomprises one or more strains of Lactobacillus having the ability tocolonize the equine intestines. The microorganisms were isolated fromthe gastric or intestinal mucosa of horses.

GB 2,167,639 discloses a process for the treatment of industrial oragricultural waste matter, such as animal protein. The process involveschopping the waste as an aqueous mass and treating the resultingmaterial with proteolytic enzymes to form a suspension, obtaining agelatinised starch content in the suspension and adding to thesuspension amylolytic enzymes and a lactic acid producing culture. Theresulting mixture is fermented to produce simple sugars and lactic acid.

U.S. Pat. No. 4,214,985 relates to sewerage treatment. The treatmentinvolves inoculating sewerage sludge with L. plantarum bacteria and acarbohydrate, such as lactose. The resulting mixture is fermented untilthe pH falls below 4.0. The thus produced composition is used as a soilextender.

JP 2007082468 is concerned with providing a microorganisms preparationfor feed. The preparation comprises particular strains of Lactobacillusplantarum and/or Bacillus subtilis. Fermented feeds may be produced byadding the microorganisms to organic wastes, such as silage grass, andfermenting.

WO 89/05849 describes the selection of lactic acid bacteria isolatedfrom the gastrointestinal tract of pigs for their ability to survive inthe environment of the gastrointestinal tract and to adhere to theepithelium of the gastrointestinal tract of the target animal. Thebacteria selected with these properties may be included in a fermentedmilk product for human consumption or in a veterinary composition forproviding to pigs for the prevention or treatment of gastrointestinaldiseases.

More recently, WO 2008/006382 discloses homofermented liquid animal feedproducts. As discussed in WO 2008/006382, the production of fermentedanimal feeds using microorganism-containing inoculants is verydifficult, often leading to the fermented feed containing pathogenicbacteria, such as Vibrio spp., Campylobacter spp., Salmonella spp., E.coli, and Staphylococcus aureus. The fermented feed may also contain ahigh content of various yeasts and moulds. It is noted that theingestion by the livestock of such inappropriately fermented feeds mayresult in morbidity and mortality. WO 2008/006382 notes that the sterilehandling of bacteria required by farmers wishing to prepare their ownfermented feed is often impossible to achieve. Further, there is apractice of using a continuous fermentation process, in which a portionof one batch of fermented feed is used as an inoculum for a subsequentfermentation batch. This leads to a gradual build up of harmful andundesirable microorganisms in the fermented feed. In an attempt toaddress these problems, WO 2008/006382 proposes a method for preparing afermented mixed feed, the method comprising: providing a liquidfermented product; providing a feed product to be fermented; combiningthe aforementioned products; and fermenting the feed product using theliquid fermented product as an inoculum. A fermented feed prepared bythis method is also described.

While proposals have been made to provide fermented feeds that areresistant to spoilage due to the growth of yeasts, moulds, bacteria andother organisms, there is still a need for an improved fermented feedthat may be stored for extended periods of time and transported, withoutsignificant spoilage.

As mentioned in WO 2008/006382, a further issue relating to feedstuffs,in particular moist or liquid feed materials, is the health andwellbeing of the livestock consuming the feeds. As noted in WO2008/006382, a poorly fermented feed may be a source of microorganismsharmful to the animals consuming the feed. More generally, animals aresusceptible to a wide range of infections arising from microorganismsthat enter and colonise the gastrointestinal (GI) tract of the animal.EP 0 955 061 addresses the issue of gastroenteric infections in pigs, inparticular porcine rotavirus, porcine coronavirus, enterotoxigenic andenteropathogenic strains of Escherichia coli, Clostridium sp.,Salmonella sp., Serpulina hyodysenteriae, Serpulina pilosicoli, Lawsoniaintracellularis, Isospora suis, and Cryptosporidium. As a solution tothe problem of gastroenteric infections in pigs, EP 0 955 061 proposesan oral product characterised by containing at least one specificantibody to the aforementioned microorganisms, derived from the eggyolks of immunized hens. It is noted in EP 0 955 061 that lactacidogenicbacteria administered to pigs can have a probiotic effect, suppressingthe propagation of the enteropathogenic or enterotoxigenic bacteria andenhance the activity of the animal's immune system. Accordingly, apreferred embodiment of EP 0 955 061 includes one or more lactic acidbacteria, such as Enterococcus spp. and Lactobacillus spp.

As discussed in EP 0 955 061, young animals are particularly susceptibleto infections of the GI tract, leading to illness and death. Ways ofimproving the health and wellbeing of finishing pigs are described by P.Brooks et al., ‘The Effect on Biological Performance and FaecalMicrobiology of Feeding Finishing Pigs on Liquid Diets Fermented withLactic Acid Bacteria’, SafePork, 2005, page 149. Brooks et al. note thatfermented liquid feeds (FLF) have been shown to reduce the incidence ofsalmonella in pigs. In particular, it was found that a lactic acidconcentration of 70 mMol/kg in the fermented feed exhibitedbacteriostatic activity with respect to Salmonella spp., but higherconcentrations of lactic acid in excess of 100 mMol/kg were needed to bebactericidal. However, Brooks et al. had found that naturalfermentations had produced unpredictable results in commercial feedunits and referred to a study that found that only 3% of commercialfermentations of wheat and barley produced more than 75 mMol/kg oflactic acid after 24 hours of fermentation. It was concluded thatfermentations to produce lactic acid in high concentrations relying onindigenous microorganisms present in the grains could not be relied uponfor commercial production of fermented feeds. Brooks et al. conductedexperiments using specific LAB to examine the effect on biologicalperformance and faecal microbiology of pigs fed diets of fermentedliquid feeds. The results showed that the pigs retained good health whenfed on the fermented liquid feed, showing no change in average dailyweight gain when fed with the FLF compared with a standard feed. Inaddition, the experiments showed that, while the fermented feedcontained lactic acid bacteria in high concentrations, the concentrationof LAB in the faeces of the pigs remained unchanged. However, analysisof the faeces for the presence of coliforms indicated that the coliformcontent was reduced in the pigs fed with the FLF diet. This in turnindicated an improvement in the health of the pig and a lower risk ofinfection and illness. It was concluded that the selection of the LABused for fermentation was important in achieving the reduction incoliforms.

As noted by Brooks et al., achieving a specific concentration of lacticacid in the fermented feed is important in achieving the beneficialeffects of the fermented feeds. Techniques for measuring theconcentrations of lactic acid in fermented feeds are described by S. J.Niven, et al., The Simultaneous Determination of Short Chain Fatty Acid,Monosaccharides and Ethanol in Fermented Liquid Pig Diets', Animal FeedScience and Technology, 117 (2004), pages 339 to 345.

The thesis of V. Demeckova, ‘Benefits of Fermented Liquid Diets for Sowsand their Piglets’, Department of Agriculture and Food, Faculty of Land,Food and Leisure, University of Plymouth, July 2003, describesexperiments conducted with liquid feed fermented with Lactobacillusplantarum to determine their effects on the antimicrobial and potentialimmunological effects on sows in late gestation periods. The resultsindicated that certain strains of Lactobacillus were both an effectiveinoculant for the preparation of fermented liquid feeds, as well asproviding probiotic activity to the sow once the fermented feed wasingested. Significantly, immunoglobulin levels in the sows' colostrumswere increased. Colostrum from sows fed fermented feed also increasedthe mitogenic activity of blood lymphocytes and enterocytes.

These factors could in turn improve the resistance of the sows and theirpiglets to pathogen challenges.

Drago, L., et al., ‘Inhibition of in vitro growth of enteropathogens bynew Lactobacillus isolates of human intestinal origin’, FEMSMicrobiology Letters, 153 (1997), pages 455 to 463, describe experimentsto isolate strains of Lactobacillus from the faeces of new born infanthumans and examine their effect in co-cultures on certain pathogenicbacteria. The experiments conducted were entirely in vitro and, whileshowing some beneficial effects of the Lactobacillus strains in reducingthe growth of certain pathogens, did not relate at all to theformulation of feeds for animals.

It would be most advantageous if a fermented liquid feed compositioncould be provided that may be prepared on a commercial scale from a widerange of raw and starting materials, that would be biopreserved andexclude potentially harmful enteropathogens. It would be furtheradvantageous if the fermented feed could provide a probiotic effect tothe animals receiving it and reduce or prevent illness of the animalsdue to infections and pathogenic challenge.

The inventors have now found that such a fermented liquid feedcomposition may be produced using one or more lactic acid bacteriapossessing certain characteristics.

Accordingly, in a first aspect, the present invention provides afermented liquid feed composition for a target animal, the feedcomposition being prepared by the fermentation of a feed substrate witha lactic acid producing bacteria, the lactic acid bacteria beingcharacterised by:

-   -   a) being viable under the conditions prevailing in the        gastrointestinal tract of the target animal;    -   b) being a bacteria capable of aggregating and/or coaggregating        with one or more pathogens; and    -   c) being able to produce upon fermentation in the feed substrate        lactic acid in an amount of at least a minimum inhibitory        concentration of lactic acid.

It has been found that a fermented feed according to the presentinvention produced by the fermentation of a feed substrate with a lacticacid producing bacteria having the characteristics set out aboveprovides significant advantages over known feeds. In particular, thefeed is able to be stored for extended periods of time and betransported without spoiling, the fermented feed being resistant to thegrowth of mould and yeasts and resistant to invasion and colonisation bybacteria potentially harmful to the target animals and other livestock.Further, the fermented feed, once consumed, provides significantprotection for the animals against infection by bacteria, in particularpathogenic bacteria likely to cause serious illness or death of theanimal. The fermented feed of the present invention achieves this byenhancing the barrier function of the upper gastrointestinal tract ofthe target animal. This advantage is particularly significant in thefeeding of newly born and newly weaned animals, and newly hatched birds.In the large scale rearing of animals, for example cattle, pigs andpoultry, there are particular challenges when young animals are removedfrom their mothers (weaned) and reared in a different environment. Theweaning process removes immunoglobulin support provided by the mother'smilk and precipitates changes to the gut ecosystem. This, in turn,leaves the young animals open to infection with a wide range ofpotentially harmful microorganisms. The high mortality rate of younganimals is at least in part due to animals succumbing to such infectionsof microorganisms. The fermented feed of the present invention reducesor eliminates this risk, by populating the gastrointestinal tract of theyoung animal with healthy, beneficial microorganisms, in turn providingprotection of the young animals against infection by pathogenicmicroorganisms.

The fermented feed of the present invention is provided for a targetanimal, which may determine such factors as the composition of the feedand the particular bacteria employed in the fermentation of the feedsubstrate. The fermented feed of the present invention may be providedfor a wide range of animals and livestock, including mammals andpoultry. Examples of target mammals include all the mammals farmed orreared, including horses, sheep, goats, pigs, cattle and deer, as wellas animals reared for fur, such as mink and the like. Examples of targetpoultry include all the birds reared and farmed on a commercial scale,including chickens, ducks, geese, quail and turkeys, as well as gamebirds, such as pheasants, partridges and the like. The fermented feedmay also be provided to farmed and ornamental fish and crustaceans.Further, the fermented feed may be provided for animals kept as domesticpets, such as dogs, cats, rabbits and the like.

In one preferred embodiment, the fermented feed of the present inventionis advantageously formulated for providing to poultry, includingchickens, quail, turkeys, geese, ducks and the like.

In a second preferred embodiment, the fermented feed of the presentinvention is advantageously formulated for providing to mammals, inparticular pigs and ruminants, such as cattle and sheep, particularlyduring the post-natal and pre-ruminant stage and veal calves, in whichruminant function my be delayed.

The fermented feed of the present invention may be provided to any ageof target animal, from newly born animals or newly hatched birds tomature adult animals. The fermented feed has been found to beparticularly advantageous when provided to newly born and newly weanedanimals or newly hatched birss, where the fermented feed provides suchadvantages as increased weight gain of the young animals and a reductionin infection with potentially harmful or pathogenic microorganisms. Thisresults in a reduction in the harmful or pathogenic microorganisms shedby the animals, in turn increasing the health of other animals beingreared in the same environment and the ultimate consumer of the animaland/or its products.

The fermented feed is prepared from a feed substrate, which isinoculated with a culture containing the lactic acid producing bacteriaand fermented. The thus inoculated and fermented substrate may itselfform the finished feed. Alternatively, the substrate, once fermented maybe added to other feed materials, in order to provide the othermaterials with the biopreservative effects.

The feed substrate may be any suitable substrate that may be consumed bythe target animals in a fermented condition. The feed substrate mayconsist of a substrate from a single or source or, alternatively maycomprise a combination of substrates.

Suitable substrates include organic materials, such as plants or plantmaterial, for example, fibrous plant material, such as grass; cerealsand grains, such as wheat, barley, maize, rice, sorghum and rye; wholecrop cereals, maize silage and corn cob meal; root crops, such aspotatoes, swedes, fodder beet, sugar beet and the like; pulses andseeds, such as beans, peas, soya bean and rapeseed (and their residues);brassiccas and the like. Further substrates include organic residues,such as materials produced as co-products or residues from dairyoperations, such as whey, curd and skimmed milk, ice cream, yoghurt,off-specification butter and cheese; from the baking and confectionaryindustry, such as biscuit meals, cereal residues, misshapen andoff-specification breads, cakes and biscuits; from the beverageindustry, such as fruit pulps, grape pulp, coffee and chocolateresidues; brewing and distilling residues; from cooking oil extraction,such as rapeseed meal, soya bean meal and olive pulp; from meat and fishslaughter and processing; or the production of bio-fuels.

To be suitable for fermentation, the feed substrate should contain waterin an amount sufficient to support fermentation with the lactic acidproducing bacteria. Preferably, the feed substrate has a water contentof at least 20% by weight, more preferably at least 30% by weight, stillmore preferably at least 40% by weight. Preferred ratios of dry feedsubstrate to water are dependant on the target species to be fed andrange from 1:0.25 to 1:4, more preferably from 1:0.4 to 1:2. Onepreferred ratio of dry feed substrate to water is about 1:1.2 forchickens and 1:2.5 for pigs. The precise water content of the feedsubstrate will be determined by such factors as the nature andcomposition of the feed substrate, the lactic acid producing bacteriabeing employed and the end use of the fermented feed and target animal.References herein to a ‘moist feed’ or ‘liquid feed’ are to a feedmaterial containing at least the minimum water content to supportfermentation of the feed by the lactic acid producing bacteria and theterms ‘moist feed’ and ‘liquid feed’ are to be understood andinterpreted accordingly.

The feed substrate may contain sufficient water to support fermentationwith the lactic acid bacteria. If not, water should be added to the feedsubstrate to achieve the water content required for fermentation.

To produce the fermented feed of the present invention, the feedsubstrate is inoculated with lactic acid producing bacteria andfermented. The lactic acid producing bacteria employed in the presentinvention are characterised by the following features:

-   -   a) The bacteria are viable under the conditions prevailing in        the gastrointestinal tract of the target animal;    -   b) The bacteria are capable of aggregating and/or co-aggregating        with one or more pathogens; and    -   c) The bacteria are capable of producing lactic acid upon        fermentation with the feed substrate to at least a minimum        inhibitory concentration in the fermented feed.

Bacteria having the three characteristics (a) to (c) give rise to theadvantageous properties of the fermented feed of the present invention.As noted above, the feed substrate is inoculated with the lactic acidproducing bacteria.

Accordingly, in a further aspect, the present invention provides aninoculant for the preparation of a fermented feed from a feed substrate,the inoculant comprising a viable culture of a lactic acid producingbacteria having the following characteristics:

-   -   a) being viable under the conditions prevailing in the        gastrointestinal tract of the target animal;    -   b) being a bacteria capable of aggregating and/or co-aggregating        with one or more pathogens; and    -   c) being able to produce upon fermentation in the feed substrate        lactic acid in an amount of at least a minimum inhibitory        concentration of lactic acid.

The inoculant may be in any suitable form and of any suitablecomposition so as to contain viable lactic acid producing bacteria forpopulating and fermenting the feed substrate. Preferred presentationsfor the inoculant are freeze dried or as a liquid culture.

The inoculant should contain the lactic acid producing bacteria in aviable form and in sufficient concentration to allow the feed substrate,once inoculated, to ferment and produce the required number of viablelactic acid producing bacteria and the required concentration of lacticacid in the fermented feed. A typical number of lactic acid producingbacteria in the inoculant is from 10⁵ to 10⁹ CFU/ml, more preferablyabout 10⁶ CFU/ml, if presented in liquid form or 10⁵ to 10⁹ CFU/g, morepreferably about 10⁶ CFU/g if presented in freeze dried form.

For example, a suitable inoculant for a substrate is 0.1% of a liquidbroth culture containing 10⁹ CFU/ml of the lactic acid producingbacteria or 0.1% of a freeze dried culture containing 10⁹ CFU/g of thelactic acid producing bacteria.

The inoculant organism may be presented in a suitable carrier tomaintain shelf life and facilitate accurate dispersion when added to thesubstrate. Such methods are known in the art and readily understood bythe person skilled in the art.

As a first characteristic, the lactic acid producing bacteria should beviable and survive in the gastrointestinal tract of the target animal.The conditions in the gastrointestinal tract of many animals are severeenough to prevent the colonisation and growth of many microorganisms. Inparticular, the upper gastrointestinal tract of many animals issufficiently acidic to prevent many species of microorganisms fromthriving and remaining viable. In order to provide the advantageousproperties of the fermented feed of the present invention, the lacticacid producing bacteria used to ferment the feeds substrate should beviable under the acidic conditions prevailing in the uppergastrointestinal tract of the target animal and the alkaline conditionsencountered in the duodenum, and should remain viable in both the smallintestines and the large intestines. In addition, in the case offermented feed intended for providing to poultry, the lactic acidproducing bacteria should also remain viable under the conditionsprevailing in the crop and proventiculus, as well as the gizzard.

The viability of the bacteria in the gastrointestinal tract may bedetermined by methods and techniques known in the art. In particular,the microbial count of the viable lactic acid bacteria in the faeces oftarget animals fed a diet containing viable lactic acid bacteria may bemeasured. Alternatively, the lactic acid bacteria count in thegastrointestinal tract of poultry fed a diet containing the bacteria maybe determined using cloacal swabs. Such methods are known in the art andreadily understood by the person skilled in the art.

As a further alternative or in addition thereto, the viability of thelactic acid producing bacteria in the gastrointestinal tract of thetarget animal may be determined in vitro, in particular by measuring thegrowth of the microorganisms under acidic conditions similar to or thesame as those prevailing in the upper gastrointestinal tract of thetarget animal. Thus, in the case of a fermented feed intended for pigs,the viability of the lactic acid producing bacteria may be determinedafter exposure to pH 2 for 2 hours followed by buffering to pH 6.8 andexposure to bile salts for 4 hours in a suitable feed substrate, torepresent conditions in the stomach and the small intestine of thetarget animal. The acidity of the large intestine is generally similarto that of the small intestine, meaning that viability in the largeintestine is most likely for all microorganisms surviving underconditions of lower pH prevailing in the stomach. Similarly, viabilityin the gastrointestinal tract of poultry may be determined bysequentially exposing the lactic acid producing bacteria in a suitablefeed substrate to a pH of from 4.4 to 4.5, as encountered in the cropand proventiculus, a pH of about 2.6, as encountered in the gizzard, andto a pH of 6.2 in the presence of bile salts as encountered in the smallintestine of the target birds. Again, conditions in the caecum areunlikely to adversely affect organisms that survive the gizzard andsmall intestine.

In order to more accurately model the conditions of the gastrointestinaltract of the target animal, it is further preferred that the in vitroexperiments described hereinbefore are conducted using the feedsubstrate of the eventual fermented feed as the growth medium for themicroorganisms. In this way, any effects produced within thegastrointestinal tract of the target animal when fed with the fermentedfeed that may alter the conditions therein and/or the viability of thelactic acid producing bacteria may be determined.

A procedure for the in vitro determination of the viability of a lacticacid producing bacteria in the gastrointestinal tract of a target animalis described in detail in Example 1 hereafter.

In a particularly preferred embodiment, the fermented feed of thepresent invention comprises lactic acid producing bacteria that havebeen demonstrated to be viable in the gastrointestinal tract of thetarget animal using the aforementioned in vitro procedure employing thefeed substrate as growth medium for the microorganisms.

As a second requirement, the lactic acid producing bacteria of thefermented feed of the present invention are aggregating bacteria, thatis the bacteria form aggregates. In addition, or alternatively, thebacteria are capable of co-aggregating with other microorganisms, inparticular microorganisms that are pathogenic to the target animal, thatis form aggregates together with the other microorganisms. Preferably,the lactic acid producing bacteria are both aggregating andco-aggregating. The ability to aggregate and/or co-aggregate may beexhibited by the lactic acid producing bacteria under the conditions inthe feed substrate during and after fermentation and/or under theconditions prevailing in the gastrointestinal tract of the targetanimal. Preferably, the bacteria are aggregating and/or co-aggregatingboth in the feed substrate and in the gastrointestinal tract of thetarget animal.

The ability of the microorganisms to aggregate in vitro gives a strongindication of their ability to adhere to the mucus layer in the gut andthe epithelial cells of the intestinal wall of the target animal and,generally, to colonise the gastrointestinal tract. This in turnincreases the resistance of the target animal to infection by exclusionof harmful or pathogenic microorganisms from attachment sites. Further,the lactic acid producing bacteria are preferably ones that arecoaggregating, that is form coaggregations with other microorganisms, inparticular harmful or pathogenic bacteria. In particular, it ispreferred that the lactic acid producing bacteria are coaggregating withstrains of Salmonella, E. Coli, and/or Clostridium. This in turnincreases the passage and clearance of the harmful bacteria from the gutlumen.

The ability of a lactic acid producing bacteria to aggregate may bedetermined by in vitro methods and techniques known in the art, forexample as described in Drago, L. et al., noted above. In particular,the bacteria may be cultured in a suitable liquid growth medium, such asMan-Rogosa-Sharpe (MRS) broth (available commercially). Bacterialaggregates may be identified as grains or particles that develop in theliquid culture medium, typically collecting at the bottom of the culturevessel under the action of gravity and leaving a clear supernatantliquid.

Similarly, the ability of the lactic acid producing bacteria tocoaggregate with other bacteria may be determined by preparing aco-culture of the lactic acid producing bacteria with one or more targetbacteria in like manner with the formation of aggregates being observedas grain-like particles that tend to settle in the culture, againleaving a clear supernatant liquid.

While the formation of aggregates and coaggregates in the bacterialcultures may be observed using the naked eye, as described above,further and more detailed information regarding the aggregating abilityof the microorganisms may be obtained by using microscopy techniques,including scanning electron microscopy (SEM).

A procedure for the identification of lactic acid bacteria that areaggregating and coaggregating is set out in Example 2 below.

As a third characteristic, the lactic acid producing bacteria of thefermented feeds of the present invention are capable of producing atleast a minimum inhibitory lactic acid concentration in the fermentedfeed. In respect of the fermented feeds of the present invention, theterm ‘minimum inhibitory lactic acid concentration’ is a reference to alactic acid producing bacteria that is capable of producing at least 150mMol of lactic acid in 24 hours upon fermentation at 30° C. in a growthmedium consisting of MRS broth containing 2% by weight glucose. It hasbeen found that lactic acid producing bacteria that are capable ofproducing this minimum concentration of lactic acid in theaforementioned test are particularly advantageous in the preparation offermented feeds and providing significant health benefits to the targetanimals provided with the feed.

The concentration of lactic acid in the culture medium may be determinedusing methods known in the art, for example the method of Niven, S. J.,et al., The simultaneous determination of short chain fatty acidmonosaccharides and ethanol in fermented liquid pig diets', Animal FeedScience and Technology, 117 (2004), (3-4), pages 339 to 345.

A procedure for identifying lactic acid producing bacteria capable ofproducing at least the minimum inhibitory lactic acid concentration isset out in Example 3 below.

More preferably, the lactic acid producing bacteria is capable ofproducing at least 200 mMols of lactic acid under the aforementionedprocedure and test conditions, still more preferably at least 250 mMolsof lactic acid. Lactic acid concentrations of at least 300 mMols, morepreferably at least 350 mMols produced under the aforementioned testconditions may also advantageously be applied.

In general, a higher concentration of lactic acid in the fermented feed,and consequently a lower pH value for the fermented feed is to bepreferred. Accordingly, preferably the pH value of the fermented feed is4.5 or lower, more preferably 4.0 or lower, still more preferably 3.5.The lower limit of pH value and, hence, the upper limit for lactic acidconcentration will be, at least in part, determined by the target animaland its ability and willingness to eat the fermented feed. As the pH islowered further, the target animals may refuse to eat the feed.

The lactic acid producing bacteria employed to prepare the fermentedfeed of the present invention may be either homofermenting orheterofermenting. Heterofermenting bacteria produce lactic acid as aproduct of their metabolism, along with other organic acids, such as,for example, acetic acid, propionic acid and butyric acid. However, ithas been found that the presence of significant quantities of theseother acid metabolites may adversely affect the taste of the fermentedfeed and/or reduce the nutritional value of the feed to the targetanimal. In contrast, homofermenting lactic acid producing bacteria areones that metabolise the feed substrate to produce lactic acid as theonly acid metabolite. Accordingly, it is preferred that the lactic acidproducing bacteria present in the fermented feed are homofermenting.

Further, the lactic acid producing bacteria used in the fermented feedof the present invention are preferably antagonistic towards pathogenscommon to the target animal. For example, in the case of fermented feedintended to be provided to poultry, it is preferred that the lactic acidproducing bacteria have antagonistic activity against one or morestrains of Salmonella, Clostridium and E. coli.

A procedure for determining the antagonistic activity of a lactic acidproducing bacteria is set out in Example 4 below.

In addition, the lactic acid producing bacteria used in the fermentedfeed of the present invention are preferably capable of adhering to theepithelial cells of the gastrointestinal tract of the target animal. Invitro methods for determining the adhesion of bacteria in this mannerare known in the art.

A procedure for determining the ability of the lactic acid producingbacteria to adhere to the epithelial cells of the target animal is setout in Example 5 below.

Suitable lactic acid producing bacteria for use in the fermented feed ofthe present invention are naturally occurring and may be isolated fromsuitable sources using techniques known in the art. Suitable sources oflactic acid producing bacteria for use in the present invention includethe gastrointestinal tract of animals and birds, including but notlimited to the gastrointestinal tract of the target animal or bird ofthe fermented feed concerned. Other sources of lactic acid producingbacteria include cereal grains, spontaneous fermentations in substrates,and the teats and other parts animals. Isolation of the lactic acidproducing bacteria may be carried out using techniques known in the art.

Lactic acid producing bacteria may be identified again using techniquesknown in the art. For example, Lactobacilli may be identified using thegram stain and catalase tests, with Lactobacilli being gram positive andcatalase negative rods.

In a further aspect, the present invention provides a method forpreparing a fermented feed composition, the method comprising fermentinga feed substrate with a lactic acid producing bacteria, the lactic acidbacteria being characterised by:

-   -   a) being viable under the conditions prevailing in the        gastrointestinal tract of the target animal;    -   b) being an aggregating bacteria and/or co-aggregating with one        or more pathogenic bacteria; and    -   c) being able to produce upon fermentation in the feed substrate        lactic acid in an amount of at least a minimum inhibitory        concentration of lactic acid.

The fermented feeds of the present invention may be prepared in anysuitable manner. Typically, the fermented feeds are prepared byinoculating the feed substrate with an inoculum containing the lacticacid producing bacteria in viable form and fermenting the feed substrateunder suitable conditions. Techniques for fermenting a feed substrateafter inoculation with a lactic acid producing bacteria are known in theart.

The feed composition being fermented contains water. If a dry feedsubstrate is being employed, water is added to the substrate. The feedcomposition being fermented preferably contains water in an amount offrom 1 to 10 parts water by weight for each part of feed substrate (drybasis), more preferably from 1 to 5 parts water, by weight. Onepreferred embodiment comprises the feed substrate and water in a weightratio of from 1:1 to 1:3, more preferably from 1:1 to 1:2, especiallyfrom 1:1 to 1:1.5.

Fermentation of the feed substrate may be conducted at any temperaturesuitable for the cultivation of the lactic acid producing bacteria. Theoptimum temperature for fermentation will depend upon the strain orstrains of bacteria being employed. Typically, the feed substrate isfermented at a temperature of from 15 to 45° C., more preferably from 30to 35° C.

The feed substrate is fermented for a sufficient period of time to allowthe lactic acid producing bacteria to produce at least a minimum lacticacid concentration of 150 mMol/l lactic acid, more preferably at least200 mMol/l, still more preferably at least 250 mMol/l. Typicalfermentation times are from 8 to 72 hours, more preferably from 8 to 24hours.

The production of lactic acid in the fermented feed may be monitored bymeasuring the pH of the feed composition, which will fall as lactic acidis produced during the fermentation process. The pH of the feedcomposition after fermentation with the lactic acid producing bacteriais preferably 4.5 or lower, more preferably 4.0 or lower.

As noted above, feeds having a low pH may be unpalatable to the targetanimals. Fermented feeds having higher concentrations of lactic acid andpH values below 3.5 may be advantageously combined with other materialsto produce a final diet, as long as the minimum inhibitory concentrationof lactic acid is maintained.

Nutrients and other components essential to the growth of the lacticacid producing bacteria may be added to the feed substrate, as required.Such nutrients and components will be known in the art.

The feed substrate is fermented to produce a concentration of lacticacid producing bacteria in the feed composition that is beneficial tothe target animals. In particular, the lactic acid bacteria present inthe feed composition after fermentation is completed should be viable insufficient numbers to colonise the gastrointestinal tract of the targetanimal and form viable colonies therein. Preferably the concentration oflactic acid producing bacteria in the fermented feed is at least 10⁶CFU/ml, more preferably from 10⁷ to 10¹⁰ CFU/ml, still more preferablyfrom 10⁹ to 10¹⁰ CFU/ml.

The feed composition and method of the present invention may employ anysuitable lactic acid producing bacteria, with the proviso that thebacteria is not harmful to the target animal. Preferred lactic acidproducing bacteria include strains of Lactobacillus and Pediococcus,with strains of Lactobacillus being particularly preferred. Particularlypreferred microorganisms of the strain Lactobacillus include strains ofLactobacillus plantarum and Lactobacillus salivarius.

Extensive work has been carried out to isolate a series of strains ofLactobacillus of particular advantage in the preparation of fermentedfeeds according to the present invention. The strains were isolated bythe general method described hereinbefore and using the detailed methoddescribed below. Each of the isolated strains exhibited all three of theproperties (a) to (c) described above, making them particularly suitablefor use in the preparation of a feed composition according to thepresent invention. Each of the isolated strains has been deposited on 11Feb., 2009, with the National Collections of Industrial and MarineBacteria Ltd., Aberdeen, Scotland (hereafter ‘NCIMB’) and accorded theNCIMB accession numbers set out below. The deposits have been madepursuant to and in satisfaction of the requirements of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Protection.

Accordingly, in a further aspect, the present invention providesbiologically pure cultures of the following microorganisms:

Lactobacillus plantarum, strain number C28, accession number NCIMB41605;

Lactobacillus salivarius ss. Salivarius, strain number MS3, accessionnumber NCIMB 41606;

Lactobacillus plantarum, strain number MS18, accession number NCIMB41607;

Lactobacillus plantarum, strain number VD23, accession number NCIMB41608;

Lactobacillus salivarius ss. Salivarius, strain number MS6, accessionnumber NCIMB 41609; and

Lactobacillus salivarius ss. Salivarius, strain number MS16, accessionnumber NCIMB 41610.

In a further aspect, the present invention provides a composition forthe preparation of a fermented feed, the composition comprising one ormore of the aforementioned microorganisms and a suitable carrier.

On a more general note, it has been found that the administration totarget animals of lactic acid producing bacteria having the followingcharacteristics:

-   -   a) being viable under the conditions prevailing in the        gastrointestinal tract of the target animal;    -   b) being an aggregating bacteria and/or co-aggregating with one        or more pathogens; and    -   c) being able to produce upon fermentation in the feed substrate        lactic acid in an amount of at least a minimum inhibitory        concentration of lactic acid;

is generally advantageous for the health and wellbeing of the targetanimal.

Accordingly, in a further aspect, the present invention provides amethod for improving the general health of a target animal, the methodcomprising administering to the animal lactic acid producing bacteriahaving the following characteristics:

-   -   a) being viable under the conditions prevailing in the        gastrointestinal tract of the target animal;    -   b) being an aggregating bacteria and/or co-aggregating with one        or more pathogens; and    -   c) being able to produce upon fermentation in the feed substrate        lactic acid in an amount of at least a minimum inhibitory        concentration of lactic acid.

The microorganisms may be administered by way of the water provided tothe animals, for example as a single dose or by continuous feeding withwater containing the microorganisms. Alternatively, the microorganismsmay be administered by way of the feed provided to the target animals,most preferably by way of a fermented feed as hereinbefore described.

The preferred lactic acid producing bacteria for general administeringto target animals are as set out above.

The lactic acid producing bacteria are preferably administered to thetarget animal as viable microorganisms, preferably in a concentration ofat least 10⁶ CFU/ml, more preferably at least 10⁷ CFU/ml, still morepreferably in a concentration of at least 10⁹ CFU/ml. If administered tothe target animal by way of its water, the minimum number ofmicroorganisms is preferably at least 10⁶ CFU/ml. If administered by wayof a fermented feed, the minimum number of lactic acid producingbacteria is preferably at least 10⁸ CFU/ml, more preferably up to 10¹⁰CFU/ml.

It has been found that providing the target animals with lactic acidproducing bacteria in this way increases the rate at which the animalincreases in weight, and improves the overall health of the animal, inparticular increasing the resistance of the animal to infection frompotentially harmful microorganisms. This reduces the level at which thetarget animals shed harmful bacteria into their environment, in turnreducing the rate of infection of other animals in the vicinity of thetarget animals. These advantages have been found to be particularlymarked when the lactic acid producing bacteria are provided to veryyoung or immature animals.

Further, the present invention provides a biologically pure culture of alactic acid producing bacteria having the following characteristics:

-   -   a) being viable under the conditions prevailing in the        gastrointestinal tract of the target animal;    -   b) being an aggregating bacteria and/or co-aggregating with one        or more pathogens; and    -   c) being able to produce upon fermentation in the feed substrate        lactic acid in an amount of at least a minimum inhibitory        concentration of lactic acid.

A composition for providing lactic acid bacteria to a target animalcomprises a viable culture of the aforementioned lactic acid producingbacteria having characteristics (a) to (c) and a suitable carrier.

The present invention will be illustrated by way of the followingspecific examples and by reference to the accompanying figures, inwhich:

FIG. 1 is a diagrammatical representation of a preferred in vitro methodof determining the viability of lactic acid producing bacteria in thegastrointestinal tract of the target animal;

FIG. 2 is a diagrammatical representation of a preferred method fordetermining the aggregating and coaggregating ability of lactic acidproducing bacteria; and

FIG. 3 is a diagrammatical representation of a preferred method fordetermining the antagonistic level of lactic acid producing bacteria.

The experiments described in the following examples were conducted usinglactic acid producing bacteria sourced, isolated and identified asfollows:

Three chickens (Hubbard breed; age 9 weeks and 2 days) were fed adlibidum on a diet of a commercially available organic growers ration,grass and clover. The chickens were humanely slaughtered and the entiregastrointestinal tract removed from each bird. Contents from the caecum,jejunum, ileum and crop were removed aseptically. In addition,epithelial cells were removed from the small intestine and the crop byscraping with a slide. All samples were diluted in 10 mlphosphate-buffered saline (PBS, ex. Oxoid, England) and plated inMan-Rogosa-Sharpe (MRS) and Rogosa agar (both ex. Oxoid, England). Thestreak method of isolation was used to obtain pure cultures from a mixedculture of bacteria. The thus isolated pure cultures were cultured for asecond time in MRS agar plates and incubated in anaerobic jars in anatmosphere containing 5% vol carbon dioxide for 72 hours.

A total of 111 lactic acid producing bacteria were isolated on MRS agar(isolation medium for Lactobacillus and Pediococcus strains) and Rogosaagar (isolation medium for Lactobacillus strains).

Gram stains and catalase tests were used to confirm that the isolateswere lactic acid producing bacteria. Isolates that were Gram positiveand catalase negative were further identified by differentialcarbohydrate metabolism using API CHL kits (ex. BioMereux, UK).

The lactic acid producing bacteria thus isolated and identified as suchwere subjected to analysis using the procedures of Examples 1 to 3 toidentify those meeting the requirements of:

-   -   a) being viable under the conditions prevailing in the        gastrointestinal tract of the target animal;    -   b) being an aggregating bacteria and/or co-aggregating with one        or more pathogens; and    -   c) being able to produce upon fermentation in the feed substrate        lactic acid in an amount of at least a minimum inhibitory        concentration of lactic acid.

In addition, the antagonistic activity of the isolated lactic acidproducing bacteria against key pathogenic bacteria was determinedfollowing the procedure set out in Example 4. Further, the ability ofthe bacteria to adhere to epithelial cells was determined using theprocedure set out in Example 5.

EXAMPLES Example 1 Determination of Viability of Lactic Acid ProducingBacteria in the Gastrointestinal Tract of the Target Animal

The viability of strains of lactic acid producing bacteria in thegastrointestinal tract of chickens was determined using the followingprocedure, which is summarised in FIG. 1:

Each strain of lactic acid producing bacteria was sprayed onto acommercially available pelleted poultry grower feed (ex. Mole ValleyFarmers, Devon, UK). Prior to spraying with the bacteria, the feed wassterilised by irradiation (25 kGy, Co⁶⁰). The composition of thepelleted feed was as follows:

TABLE 1 Component Composition (% weight on a dry basis) Barley 4.97Wheat 55.00 Sunflower 9.00 Wheatfeed 20.00 Argentinean Soya 0.50 NGMHi-pro Soya 7.10 Limestone Flour 1.50 Di-calcium Phosphate 0.63 Salt0.25 Poultry GP Mins 1.00 Methionine 0.05

A sample of the inoculated feed was added to a flask, diluted with theaddition of distilled water and heated in a water bath to 41.4° C., torepresent the temperature within the gastrointestinal tract of achicken. The pH of the sample of the feed composition was adjustedsuccessively by the addition of HCl (aq; 1M) to adjust the pH in theflask to correspond to the pH found at the successive stages in thedigestive tract of poultry: pH 4.4 to 4.5 to correspond to the crop andproventiculus; pH 2.6 to correspond to the gizzard; and pH 6.2corresponding to the small intestine. The sample was incubated at eachpH for a period of time corresponding to the time digesta take to passthrough the corresponding portion of the gastrointestinal tract: 45minutes for the crop and proventiculus; 90 minutes for the gizzard; and90 minutes for the small intestine.

HCl (aq; 1M) was added periodically to each sample throughout theincubation period, in order to maintain the pH at the appropriate leveland counteract the normal buffering action of the feed components.

Samples (1 ml) of the solution in the flask was removed immediatelybefore the pH was adjusted at each stage in the incubation, diluted withsterile peptone water (9 ml) and 10 fold serial dilutions were prepared.100 μl of each dilution were spread over MRS agar using aseptictechniques and the plates incubated at 37° C. for 24 hours, after whichthe plates were counted. The viability of the microorganisms wascalculated as the percent of organisms surviving passage through thesimulated GI tract.

Example 2 Determination of Lactic Acid Bacteria that are Aggregating andCoaggregating

The ability of the lactic acid bacteria strains to aggregate and formcoaggregates with other bacteria was determined using the followingprocedure, as illustrated in FIG. 2:

Lactic acid producing bacteria were grown overnight in MRS broth (exOxoid) at 37° C. in an atmosphere of 5% vol carbon dioxide. Thereafter,the cultures were centrifuged for 10 minutes at 10000 times gravity andwashed three times with sterile distilled water. The thus washedmaterial was resuspended in the same volume of phosphate-buffered saline(PBS) at a concentration of 10⁹ CFU/ml at a pH of 6.0 and incubated atroom temperature.

Autoaggregation was determined to occur when clearly visible, sand-likeparticles were formed by the aggregated cells and gravitated to thebottom of the tubes within 2 hours.

In addition, the ability of the lactic acid producing bacteria toco-aggregate with other bacteria was determined by the followingprocedure:

The lactic acid producing bacteria were grown at 37° C. in MRS broth for24 hours in an atmosphere containing 5% vol carbon dioxide. Salmonellaspp. and E. coli were grown at 37° C. in nutrient for 24 hours in anatmosphere containing 5% vol carbon dioxide. Further, Clostridiumperfringens were grown in clostridial broth for 24 hours under anaerobicconditions at 37° C. The following day, each culture was centrifuged for10 minutes at 10000 times gravity and washed three times with steriledistilled water. The pathogenic cultures were resuspended inphosphate-buffered saline (PBS) to the same initial volume at aconcentration of 10⁹ CFU/ml (ph 6.0) and incubated at room temperaturein the presence of 10% vol freshly prepared filter-sterilised culture ofthe lactic acid producing bacteria supernatant liquid, at a total liquidvolume of 1 ml. Coaggregation was taken as positive when clearlyvisible, sand-like particles formed by aggregated cells settled to thebottom of the vessel under gravity, leaving a clear supernatant liquidwithin a period of 2 hours.

Co-aggregation of the lactic acid producing bacteria with otherpotentially pathogenic microorganisms was tested in similar manner andconfirmed using scanning electron microscopy.

Example 3 Determination of Lactic Acid Producing Bacteria Capable ofProducing at Least the Minimum Inhibitory Lactic Acid Concentration

The ability of the lactic acid producing bacteria strains to producelactic acid to at least the minimum inhibitory concentration wasdetermined using the following procedure:

Lactic acid producing strains were grown in MRS broth for 24 hrs at 30°C. Ten ml aliquots of fresh MRS broth were inoculated with 0.1 ml of the24 hr broth culture and incubated at 30° C. Subsamples of 1.0 ml weretaken after 12, 24 and 48 hours for lactic acid analysis by highperformance liquid chromatography according to the method of Niven etal. Standard MRS broth contains 2% glucose as a carbohydrate sourcegiving a maximum lactic acid yield of 220 mMol/L.

Example 4 Determination of Antagonistic Activity of Lactic AcidProducing Bacteria

The antagonistic activity of the lactic acid producing bacteria strainswith respect to pathogenic microorganisms was determined using thefollowing procedure, as illustrated in FIG. 3:

The lactic acid producing bacteria were grown in MRS broth (ex. Oxoid,CM0359) at a temperature of 37° C. for 24 hours under anaerobicconditions. At the end of this period, samples of the bacteria werespotted onto MRS agar plates (ex. Oxoid) using a sterile cotton swab andincubated at 37° C. for a further 24 hours, again under anaerobicconditions, to allow colonies to develop. Nutrient agar containingapproximately 10⁷ CFU/ml of each of five pathogenic bacteria Salmonellaenteric Enteritidis (3 strains); Salmonella enteric Typhimurium (1strain); and Escherichia coli (1 strain) was poured on the agar plateand the plate incubated for a further 24 hours at a temperature of 37°C. Nutrient agar containing approximately 10⁷ CFU/ml of Clostridiumperfringens (1 strain) was poured on a second plate and incubated underanaerobic conditions at 39° C. for a further 48 hours.

At the end of the incubation periods, the plates were checked visuallyfor inhibition zones around the Lactobacilli spots and the radius of theinhibition zone was recorded. A measure of the antagonistic activity ofthe subject lactic acid producing bacteria against the target pathogensmay be obtained by measuring the radius of the inhibition zone aroundthe lactic acid producing bacteria spot. A radius of from 1 to 2 cmindicated a high level of antagonistic activity.

Example 5 Determination of Adherence of Lactic Acid Producing Bacteriato Epithelial Cells

An experiment was conducted to determine the ability of strains oflactic acid producing bacteria to adhere to epithelial cells oforganically farmed chickens using the following procedure.

The chickens were humanely slaughtered and ileal epithelial cells wereremoved by scraping the epithelium with a microscope slide. The cellsthus removed were suspended in PBS and examined to ensure that they werefree from any adherent bacteria. A haemocytometer was used to determinethe number of cells.

Selected Lactobacilli were cultured overnight in MRS broth to givebacterial count of 10⁹ CFU/ml, and resuspended in PBS to give a celldensity of 10⁸ CFU/ml. 100 μl of the Lactobacillus suspension was addedto 400 μl of the epithelial cell suspension and the mixture incubatedfor 30 minutes at 37° C. with shaking. Adhesion of the Lactobacilluscells to the epithelial cells was observed using a phase contrastmicroscope by counting the number of bacterial cells adhered toepithelial cells selected at random from the resulting suspension.

Example 6

An experiment was conducted to determine the benefits of treatingchickens with strains of the lactic acid-producing microorganismLactobacillus salivarius exhibiting the characteristics of a) beingviable in the gastrointestinal tract of chickens (determined as outlinedin Example 1); b) aggregating and co-aggregating with at least one orthe following pathogens: Salmonella, E. coli, or Clostridia (determinedas outlined in Example 2); and c) producing at least a minimuminhibitory concentration of lactic acid (determined as outlined inExample 3). In addition, the microorganisms were determined to beantagonistic to strains of Salmonella, Clostridium and E. coli using themethod set out in Example 4. Using the procedure set out in Example 5,the lactic acid-producing bacteria were also determined to be highlyadherent to chicken epithelial cells.

The strain of Lactobacillus salivarius employed was strain C28 referredto above.

Throughout the experiment, the birds were fed on a diet of clean waterand a commercially available feed (Saracen Chick Crumbs, ex. J&W Attlee,Dorking, England). The feed had a moisture content of 14.0 wt %, withthe composition, on a dry basis, as set out in Table II.

TABLE II Composition of Feed Component Composition (% wt on a dry basis)Wheat 54.5 Hipro Soya 16.7 Barley 10.0 Minerals 2.7 Peas 2.5 Fishmeal1.5 Vegetable fat 1.2 Vitamins 0.75 Methionine 0.13 Lysine 0.03

102 specific pathogen-free chickens were randomly allotted to six groupsof 17, with each group being treated as follows:

Group I: birds fed clean water and feed according to Table I.

Group II: birds treated by oral gavage at age 1 day with an aqueousmedium containing Lactobacillus salivarius in a concentration of 10⁷cfu/ml. Thereafter, the birds were fed as for Group I.

Group III: birds fed water containing 10⁷ cfu/ml Lactobacillussalivarius and feed according to Table I from age 1 day.

Group IV: birds fed water containing 10⁷ cfu/ml Lactobacillus salivariusand feed according to Table I from age 7 days.

Group V: birds fed clean water and fermented wet mash from age 1 day.The fermented wet mash was prepared by inoculating a mixture of the feedof Table I and water (ratio of feed to water of 1:1.2) withLactobacillus salivarius and fermenting for 24 hours at 30° C. to obtaina microorganism concentration of about 10⁹ cfu/nil. The feed had a meanpH before fermentation with Lactobacillus salivarius of 5.94 and a meanpH after fermentation of 4.42.

Group VI: birds fed clean water and fermented wet mash from age 7 days.The fermented wet mash was prepared by inoculating a mixture of the feedof Table I and water (ratio of feed to water of 1:1.2) withLactobacillus salivarius and fermenting for 24 hours at 30° C. to obtaina microorganism concentration of about 10⁹ cfu/ml. The feed had a meanpH before fermentation with Lactobacillus salivarius of 5.94 and a meanpH after fermentation of 4.39.

Weight of Feed Consumed by Birds

The experiment was conducted for a period of six weeks. As a firstindicator of the health of the birds, the weight of feed being consumedby the birds in each group was monitored. The average daily feedconsumption of the birds in each group during weeks 3 to 6 of theexperiment is set out in Table 2.

TABLE 2 Average Daily Feed Consumption of Birds (g/bird/day on a dryfeed basis) GROUP Week 3 Week 4 Week 5 Week 6 I 44 49 59 59 II 43 49 6175 III 43 47 52 52 IV 47 57 66 74 V 75 124 136 151 VI 77 121 132 151

As shown in Table 2, birds in Groups V and VI provided with thefermented feeds consumed significantly higher quantities of feed thanthe birds in other groups, indicative of a higher general level ofhealth for the birds in Groups V and VI. In general, the birds providedwith the lactic acid producing bacteria, whether by way of water orfermented feed, consumed larger quantities of feed, compared with thecontrol group I.

Weight Gained by Birds

In addition to the quantity of feed consumed, the average weight gainfor the birds in each group was measured. The average daily weight gainof the birds in weeks 3 to 5 of the experiment is shown in Table 3.

TABLE 3 Average Daily Weight Gain (g/bird/day) GROUP Week 3 Week 4 Week5 I 12.9 16.1 17.5 II 13.1 16.7 16.9 III 13.8 16.8 18.1 IV 14.2 16.218.7 V 15.2 16.0 22.1 VI 14.2 16.5 18.4

As shown in Table 3, the average daily weight gain of the birds treatedwith Lactobacillus salivarius was generally higher than that of theuntreated birds. In particular, the initial weight gain of all birdsprovided with the lactic acid producing bacteria was significantlyhigher than that of the control group I. Further, the birds in Groups Vand VI fed on fermented feed exhibited significantly higher weight gain,in particular in week 5 of the experiment.

Salmonella Shedding and Infection of Birds

At the start of the experiment, a random sample of birds from each groupwas cloacally swabbed and their faecal contents analysed to confirm noinfection by strains of Salmonella.

To determine the effectiveness of the treatment with Lactobacillussalivarius in preventing infection of the birds by other microorganisms,all birds in each group were challenged with a strain of Salmonellaaccording to the following procedure:

At 15 days of age, all birds were dosed with Salmonella typhimurium byoral gavage using a dosing catheter to administer an aqueous mediumcontaining 10⁶ cfu/ml of Salmonella microorganisms. The Salmonellaorganisms employed were a nalidixic acid resistant derivative (SL1344nal^(r); ex. Veterinary Laboratories Agency (VLA), Weybridge, UK).Immediately prior to dosing with Salmonella, the birds were dosed inlike manner with a solution of sodium bicarbonate, so as toinstantaneously neutralise the acidity in the crop of the bird. In thisway, the barrier imposed in the upper gut of the birds by acids in thecrop was removed, permitting access of the introduced Salmonella to thelower gut environment.

Cloacal swabs were taken from the birds immediately before challenge andat least twice a week after challenge for a period of 4 weeks. Thecontent of Salmonella typhimurium in the swabbed material wasdetermined. In addition, the Salmonella-content of the bird litter wasdetermined for each group. For each group, the percentage of birds thatwere found not to be shedding salmonella was determined. The results areset out in Table IV.

TABLE IV Salmonella shedding PERCENTAGE OF BIRDS NOT GROUP SHEDDINGSALMONELLA I 8 II 14 III 19.5 IV 23 V 64 VI 82

From Table IV it can be seen that, in general, administering lactic acidproducing bacteria to the birds significantly reduced the tendency ofthe birds to shed Salmonella into their environment. The reduction inSalmonella of the birds provided with the fermented feed, that is GroupsV and VI, is particularly marked.

Further, throughout the duration of the experiment, it was found that atall times, the Salmonella shedding exhibited by the birds wasconsistently and significantly lower in the groups fed with thefermented feeds, that is Groups V and VI.

To determine the level of infection of the birds in each group, twopost-mortem enumerations of Salmonella typhimurium infestations of thebirds were carried out, one at 4 weeks of age and one at 6 weeks of age.The method employed was as follows:

The birds were euthanized by cervical dislocation. Their liver, spleen,ileum and caeca were removed aseptically and placed in sterile PBS.Samples of each tissue thus collected were weighed, homogenised andsubjected to serial 10 fold dilutions in PBS (0.1M; pH 7.2). The viablecount of Salmonella typhimurium in each homogenate was determined byplating drops of the dilutions on BGA supplemented with nalidixic acid(15 μg/ml). 1.0 ml of residual homogenate was added to 10 ml Seleniteenrichment broth, incubated for 24 hours at 37° C., and thereaftersubcultured on BGA supplemented with nalidixic acid. Viable counts ofbacteria were determined by plating on MRS agar and incubating inanaerobic jars for 48 hours at 37° C.

The results of the first and second post mortem tests are set out inTables V and VI respectively.

TABLE V Salmonella typhimurium counts for first post mortem test CAECUMILEUM SPLEEN LIVER (log₁₀ CFU/g (log₁₀ CFU/g (log₁₀ CFU/g (log₁₀ CFU/gGROUP tissue) tissue) tissue) tissue) I 5.7 4.3 2.5 2.1 II 6.1 2.7 4.02.2 III 5.6 2.2 3.8 1.6 IV 5.2 2.0 4.4 3.3 V 2.2 0.8 0.6 0.4 VI 2.1 1.01.7 0.8

TABLE VI Salmonella typhimurium counts for second post mortem testCAECUM ILEUM SPLEEN LIVER (log₁₀ CFU/g (log₁₀ CFU/g (log₁₀ CFU/g (log₁₀CFU/g GROUP tissue) tissue) tissue) tissue) I 2.3 2.3 0.7 Nd II 4.1 2.51.9 1.8 III 2.3 1.0 Nd 0.4 IV 2.7 Nd Nd 0.2 V 1.1 Nd nd 0.7 VI 1.0 0.30.5 0.7 Nd = not detected

From Tables V and VI it can be seen that, in general, providing thebirds with lactic acid producing bacteria significantly reduced thecount of Salmonella typhimuriam in the birds. The reduction in the countof Salmonella typhimuriam was most notable in the birds in Groups V andVI fed with the fermented feeds.

Lactobacillus Salivarius Colonisation of Birds

For the birds euthanized in the tests described above, the count ofLactobacillus salivarius in the caecum and ileum of the birds was alsodetermined, using the general procedure outlined above, in order todetermine the efficiency of the lactic acid producing bacteria incolonising the gastrointestinal tracts of the birds. The results are setout in Table VII.

TABLE VII Lactobacillus salivarius in GI tract of birds CAECUM PostILEUM Mortem Post Post Post 1 Mortem 2 Mean Mortem 1 Mortem 2 Mean(log₁₀ (log₁₀ (log₁₀ (log₁₀ (log₁₀ (log₁₀ CFU/g CFU/g CFU/g CFU/g CFU/gCFU/g GROUP tissue) tissue) tissue) tissue) tissue) tissue) I 7.5 8.37.9 9.9 8.3 8.7 II 8.9 8.4 8.6 9.4 8.8 9.1 III 8.9 8.3 8.6 9.1 8.8 9.0IV 8.8 8.4 8.6 9.1 8.1 8.6 V 9.6 9.5 9.5 9.7 9.2 9.5 VI 9.5 9.6 9.5 9.79.2 9.5

As shown in Table VII, the provision of the lactic acid producingbacteria to the birds in the water and the feed significantly increasedthe degree of colonisation of the gastrointestinal tract by thebacteria. The fermented feeds provided to the birds in Groups V and VIwere particularly effective in increasing the concentration of lacticacid producing bacteria in the gastrointestinal tract of the birds.

As a general result, the fermented feeds were particularly effective inreducing the colonisation of the birds by Salmonella. This is ofsignificant advantage in the production of foodstuffs for humans, werethe prime concern of food producers is the elimination of food borepathogens, such as Salmonella from food animals and their products.Surprisingly, it was found that improved resistance to colonisation withSalmonella was achieved by providing the birds with fermented feed onlyfrom the age of 7 days, and not from age 1 day.

1-39. (canceled)
 40. A fermented liquid feed composition for a targetanimal, the feed composition being prepared by the fermentation of afeed substrate with a lactic acid producing bacteria, the lactic acidbacteria being characterised by: a) being viable under the conditionsprevailing in the gastrointestinal tract of the target animal; b) beinga bacteria capable of aggregating and/or co-aggregating with one or morepathogens; and c) being able to produce upon fermentation in the feedsubstrate lactic acid in an amount of at least a minimum inhibitoryconcentration of lactic acid.
 41. The fermented liquid feed compositionaccording to claim 40, wherein the target animal is poultry or mammals.42. The fermented liquid feed composition according to claim 41, whereinthe target animal is chickens or pigs.
 43. The fermented liquid feedcomposition according to claim 40, wherein the substrate is selectedfrom the group consisting of plants, plant material, and organicresidue.
 44. The fermented liquid feed composition according to claim43, wherein the plant material is selected from the group consisting ofgrass; cereals and grains, such as wheat, barley, maize, rice, sorghumand rye; root crops, such as potatoes, swedes, fodder beet, sugar beetand the like; pulses and seeds, such as beans, peas, soya bean rapeseed;brassiccas and the like.
 45. The fermented liquid feed compositionaccording to claim 43, wherein the organic residue comprises co-productsor remnants from the group consisting of dairy operations, such as whey,curd, skimmed milk, ice cream, yoghurt, butter and cheese; the bakingand confectionary industry; the beverage industry; brewing anddistilling; the extraction of cooking oil; meat and fish slaughter andprocessing; and the production of bio-fuels.
 46. The fermented liquidfeed composition according to claim 40, wherein the feed substrate has awater content of at least 20% by weight.
 47. The fermented liquid feedcomposition according to claim 40, wherein the ratio of dry feedsubstrate to water are from 1:0.25 to 1:4.
 48. The fermented liquid feedcomposition according to claim 40, wherein the lactic acid producingbacteria are viable under a plurality of conditions of pH correspondingto a plurality of locations in the gastrointestinal tract of the targetanimal.
 49. The fermented liquid feed composition according to claim 40,wherein the lactic acid producing bacteria are proven viable in an invitro experiment modelling the conditions in the gastrointestinal tractof the target animal.
 50. The fermented liquid feed compositionaccording to claim 49, wherein the in vitro experiment employs the feedsubstrate as the growth medium for the lactic acid producing bacteria.51. The fermented liquid feed composition according to claim 40, whereinthe lactic acid producing bacteria are coaggregating.
 52. The fermentedliquid feed composition according to claim 51, wherein the lactic acidproducing bacteria are coaggregating with respect to bacteria that areharmful or pathogens to the target animal.
 53. The fermented liquid feedcomposition according to claim 52, wherein the lactic acid producingbacteria are coaggregating with strains of Salmonella, E. Coli, and/orClostridia.
 54. The fermented liquid feed composition according to claim40, wherein the lactic acid producing bacteria are capable of producingat least 200 mMol of lactic acid in 48 hours upon fermentation at 30° C.in a growth medium consisting of MRS broth with 2% by weight glucose.55. The fermented liquid feed composition according to claim 54, whereinthe lactic acid producing bacteria produces at least 250 mMols of lacticacid under the said conditions.
 56. The fermented liquid feedcomposition according to claim 40, having a pH of 4.5 or lower.
 57. Thefermented liquid feed composition according to claim 40, wherein thelactic acid producing bacteria is homofermenting.
 58. The fermentedliquid feed composition according to claim 40, wherein the lactic acidproducing bacteria is antagonistic against one or more pathogens commonin the target animal.
 59. The fermented liquid feed compositionaccording to claim 40, wherein the concentration of lactic acidproducing bacteria in the fermented feed is at least 10⁶ CFU/ml.
 60. Thefermented liquid feed composition according to claim 40, wherein thelactic acid producing bacteria comprise strains of bacteria selectedfrom the group consisting of Lactobacillus and Pediococcus.
 61. Thefermented liquid feed composition according to claim 60, wherein thelactic acid producing bacteria comprises a strain of Lactobacillusselected from the species of Lactobacillus plantarum and Lactobacillussalivarius.
 62. The fermented liquid feed composition according to claim61, wherein the lactic acid producing bacteria are selected from thegroup consisting of: Lactobacillus plantarum, strain number C28,accession number NCIMB 41605; Lactobacillus salivarius ss. Salivarius,strain number MS3, accession number NCIMB 41606; Lactobacillusplantarum, strain number MS18, accession number NCIMB 41607;Lactobacillus plantarum, strain number VD23, accession number NCIMB41608; Lactobacillus salivarius ss. Salivarius, strain number MS6,accession number NCIMB 41609; and Lactobacillus salivarius ss.Salivarius, strain number MS16, accession number NCIMB
 41610. 63. Aninoculant for the preparation of a fermented feed from a feed substrate,the inoculant comprising a viable culture of a lactic acid producingbacteria having the following characteristics: a) being viable under theconditions prevailing in the gastrointestinal tract of the targetanimal; b) being an aggregating bacteria and/or co-aggregating with oneor more pathogens; and c) being able to produce upon fermentation in thefeed substrate lactic acid in an amount of at least a minimum inhibitoryconcentration of lactic acid.
 64. The inoculant according to claim 63,wherein the concentration of lactic acid producing bacteria in theinoculant is from 10⁵ to 10⁹ CFU/ml.
 65. The inoculant according toclaim 63, wherein the lactic acid producing bacteria comprise strains ofbacteria selected from the group consisting of Lactobacillus andPediococcus.
 66. The inoculant according to claim 65, wherein the lacticacid producing bacteria comprises a strain of Lactobacillus selectedfrom the species of Lactobacillus plantarum and Lactobacillussalivarius.
 67. The inoculant according to claim 66, wherein the lacticacid producing bacteria are selected from the group consisting of:Lactobacillus plantarum, strain number C28, accession number NCIMB41605; Lactobacillus salivarius ss. Salivarius, strain number MS3,accession number NCIMB 41606; Lactobacillus plantarum, strain numberMS18, accession number NCIMB 41607; Lactobacillus plantarum, strainnumber VD23, accession number NCIMB 41608; Lactobacillus salivarius ss.Salivarius, strain number MS6, accession number NCIMB 41609; andLactobacillus salivarius ss. Salivarius, strain number MS16, accessionnumber NCIMB
 41610. 68. A method for preparing a fermented feedcomposition, the method comprising fermenting a feed substrate with alactic acid producing bacteria, the lactic acid bacteria beingcharacterised by: a) being viable under the conditions prevailing in thegastrointestinal tract of the target animal; b) being an aggregatingbacteria and/or co-aggregating with one or more pathogens; and c) beingable to produce upon fermentation in the feed substrate lactic acid inan amount of at least a minimum inhibitory concentration of lactic acid.69. The method according to claim 68, wherein the lactic acid producingbacteria comprise strains of bacteria selected from the group consistingof Lactobacillus and/or Pediococcus.
 70. The method according to claim69, wherein the lactic acid producing bacteria comprises a strain ofLactobacillus selected from the species of Lactobacillus plantarum andLactobacillus salivarius.
 71. The method according to claim 70, whereinthe lactic acid producing bacteria are selected from the groupconsisting of Lactobacillus plantarum, strain number C28, accessionnumber NCIMB 41605; Lactobacillus salivarius ss. Salivarius, strainnumber MS3, accession number NCIMB 41606; Lactobacillus plantarum,strain number MS18, accession number NCIMB 41607; Lactobacillusplantarum, strain number VD23, accession number NCIMB 41608;Lactobacillus salivarius ss. Salivarius, strain number MS6, accessionnumber NCIMB 41609; and Lactobacillus salivarius ss. Salivarius, strainnumber MS16, accession number NCIMB
 41610. 72. A biologically pureculture of a microorganism selected from the group consisting of:Lactobacillus plantarum, strain number C28, accession number NCIMB41605; Lactobacillus salivarius ss. Salivarius, strain number MS3,accession number NCIMB 41606; Lactobacillus plantarum, strain numberMS18, accession number NCIMB 41607; Lactobacillus plantarum, strainnumber VD23, accession number NCIMB 41608; Lactobacillus salivarius ss.Salivarius, strain number MS6, accession number NCIMB 41609; andLactobacillus salivarius ss. Salivarius, strain number MS16, accessionnumber NCIMB
 41610. 73. A method for improving the general health of atarget animal, the method comprising administering to the animal lacticacid producing bacteria having the following characteristics: a) beingviable under the conditions prevailing in the gastrointestinal tract ofthe target animal; b) being an aggregating bacteria and/orco-aggregating with one or more pathogens; and c) being able to produceupon fermentation in the feed substrate lactic acid in an amount of atleast a minimum inhibitory concentration of lactic acid.
 74. The methodaccording to claim 73, wherein the lactic acid producing bacteria areadministered by means selected from the group consisting of the waterprovided to the animal and the animal feed.
 75. The method according toclaim 73, wherein the lactic acid producing bacteria are selected fromthe group consisting of Lactobacillus plantarum, strain number C28,accession number NCIMB 41605; Lactobacillus salivarius ss. Salivarius,strain number MS3, accession number NCIMB 41606; Lactobacillusplantarum, strain number MS18, accession number NCIMB 41607;Lactobacillus plantarum, strain number VD23, accession number NCIMB41608; Lactobacillus salivarius ss. Salivarius, strain number MS6,accession number NCIMB 41609; and Lactobacillus salivarius ss.Salivarius, strain number MS16, accession number NCIMB 41610.